Angiogenesis, the process of formation of new capillaries from preexisting blood vessels, is essential for the proper organ development and tissue repair (1). However, uncontrollable angiogenesis may lead to pathologies such as chronic inflammation (2), diabetic retinopathy (3), rheumatoid arthritis (4), and growth of solid tumors (5). Our long-term goals are to define the role of human alkaline phytoceramidase (haPHC) in regulating angiogenesis and to develop this concept into a new strategy to treat angiogenesis-related diseases by targeting this enzyme, haPHC, a novel enzyme which the PI identified recently, cleaves phytoceramide to generate phytosphingosine (PHS), which is in turn phosphorylated to generate phytosphingosine-1-P (PHS-1-P) through the action of sphingoid base kinases (6). Our additional preliminary data demonstrate that 1) PHS-1-P protects human umbilical vein endothelial cells (HUVEC) from apoptosis induced by serum starvation and induces migration of HUVEC; 2) the levels of PHS-1-P are regulated by haPHC through controlling the generation of its precursor, PHS from phytoceramide; 3) haPHC mRNA is highly expressed in placenta in which angiogenesis occurs actively; and 4) down-regulation of haPHC by antisense oligodeoxynucleotide (ODN) suppresses growth of HUVEC. These results suggest the hypothesis that haPHC regulates angiogenesis by regulating the levels of the potential angiogenic stimulator, PHS-1-P. Specific aim 1: to determine the role of the human alkaline phytoceramidase in growth and survival of human umbilical vein endothelial cells. Our hypothesis is that haPHC regulates growth and survival of endothelial cells by controlling the generation of PHS-1-P. To test this hypothesis, we will determine 1) whether up-regulation of haPHC, by adenovirus-mediated transfection of sense cDNA, elevates the levels of PHS-1-P in HUVEC and results in cell proliferation whereas down-regulation of haPHC by antisense cDNA transfection reduces the levels of PHS-1-P and results in apoptosis and/or growth suppression of HUVEC; and 2) whether apoptosis and/or growth inhibition induced by the downregulation of haPHC is alleviated or suppressed by exogenous PHS-1-P. We expect that downregulation of haPHC will lower the level of PHS- 1-P and induce growth suppression and apoptosis of HUVEC, while exogenous PHS-1-P will suppress these effects of haPHC down-regulation. Specific aim 2: to determine mechanism of the human alkaline phytoceramidase action. In our preliminary studies, we demonstrate that haPHC 1) is highly expressed in placenta and heart and localized to the Golgi apparatus and endoplasmic reticulum; 2) hydrolyzes NBD-Clz-phytoceramide preferentially in vitro, and 3) is activated by Ca2+,but inhibited by sphingosine (6). These results support the hypothesis that haPHC has very restricted substrate specificity; its action is regulated by second messengers; and its expression is compartment and tissue specific. To test this hypothesis, we will purify haPHC and determine its substrate specificity using different phytoceramide analogs, effects of cations and lipids on its activity, its cellular localization, and tissue specific expression. We expect that the purified haPHC 1) prefers phytoceramide with a specific acyl chain as its endogenous substrate, 2) is activated by a physiologic concentration of Ca2+, but inhibited by a low concentration of sphingosine, 3) is localized to the Golgi apparatus and ER, 4) is highly expressed in tissues where angiogenesis actively occurs. Specific aim 3: to determine the role of the alkaline phytoceramidase in angiogenesis. Our hypothesis is that haPHC regulates developmental angiogenesis and vascularogenesis by regulating the generation of PHS-1-P. To test this hypothesis, we will disrupt the mouse alkaline phytoceramidase (maPHC) gene and analyze developmental angiogenesis and vascularogenesis in maPHC null versus wild type mice. We expect that the mice lacking maPHC will have insufficient angiogenesis, which may lead to abnormalities in embryonic development. These studies should validate our hypothesis that haPHC has an important role in angiogenesis in a definitive way.